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Evaluation of procedures for detaching particle-associated microbes from forage and concentrate incubated in Rusitec fermenters: Efficiency of recovery and representativeness of microbial isolates

Departamento de Producción Animal, Universidad de León, 24007 León, Spain

mdcart{at}unileon.es

Abstract

Three detachment procedures (DP) were evaluated for their ability to remove particle-associated microbes (PAM) from digesta in Rusitec fermenters fed a 30:70 alfalfa hay:concentrate diet. Forage and concentrate were incubated in separate nylon bags, and incubation residues were treated independently. Microbial biomass was labeled with ¹⁵NH₄Cl. Treatments were: 1) MET: residues were incubated at 38ºC for 15 min with saline solution (0.9% NaCl) containing 0.1% methylcellulose with continuous shaking; 2) STO: residues were mixed with cold saline solution and homogenized with a stomacher for 5 min at 230 revolutions/min; 3) FRE: residues were immediately frozen at -20ºC for 72 h, thawed at 4ºC, mixed with saline solution and subjected to STO procedure. Common to all treatments was storing at 4ºC for 24 h after the treatment, homogenization, filtration, and resuspension of residues 2 times in the treatment solutions. Microbial pellets were obtained by centrifugation, and microbial removal was estimated indirectly by measuring removal of ¹⁵N. The PCR-single-stranded conformation polymorphism analysis of the 16S ribosomal DNA was used to analyze the similarity between microbial communities attached to the substrate and those in the pellet obtained after each DP. There were no feed x DP interactions (P = 0.16 to 0.96) for any variable, except for N content in microbial pellets (P = 0.02). Both detaching efficiency (P = 0.004) and total recovery (P = 0.01) were affected by DP, with STO showing the greatest values (mean values across substrates of 64.1% for detaching efficiency and 58.3% for total recovery) and MET the lowest ones (57.0 and 51.8%). Similarity index between the microbes attached to substrates and those in the pellets were affected (P = 0.02) by DP, with MET showing greater (P < 0.02) values (84.0 and 86.4% for forage and concentrate, respectively) than FRE (72.5 and 67.8%) and STO having intermediate values (77.1 and 82.4%). There were no differences (P = 0.70) among PAM pellets in their N content, but MET pellets had greater (P < 0.05) ¹⁵N enrichments than those obtained by STO and FRE. Although STO was the most effective method to detach ruminal microbes from both concentrate and forage, MET produced pellets with the greater similarity to the microbial communities attached to the substrates and therefore could be considered the most appropriate DP method for treating digesta from Rusitec fermenters.

Key Words: detachment • ¹⁵N • ruminal microbes • microbial diversity

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